Bisphosphonate compounds and methods for bone resorption diseases, cancer, bone pain, immune disorders, and infectious diseases

ABSTRACT

Bisphosphonate compounds and related methods of making and using are disclosed, including pyridinium-1-yl, quinolinium-1-yl, and related compounds. The activity of compounds is disclosed in the context of functional assays such as  Leishmania major  farnesyl diphosphate synthase (FPPS) inhibition,  Dictyostelium discoideum  growth inhibition, human gamma delta T cell activation, and bone resorption. The applicability of bisphosphonate compounds in the context of parasitic infections, for example against trypanosomes, is disclosed. Further potential applications of the invention are disclosed regarding the treatment of one or more conditions such as bone resorption disorders, cancer, bone pain, infectious diseases, and in immunotherapy.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/245,612 filed Oct. 7, 2005, which claims the benefit of U.S.Provisional Patent Application 60/617,108 by Sanders et al., filed Oct.8, 2004; each of which is incorporated by reference in entirety to theextent not inconsistent herewith.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made, at least in part, with government support underGrant Nos. GM50694 and GM65307 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Nitrogen-containing bisphosphonates such as pamidronate (Aredia®) C1;alendronate (Fosamax®) C2; risedronate (Actonel®) C3; and zoledronate(Zometa®) C4; shown in their zwitterionic forms in FIG. 1, represent animportant class of drugs, currently used to treat osteoporosis, Paget'sdisease and hypercalcemia due to malignancy. See references 1-4. Thesecompounds function primarily by inhibiting the enzyme farnesyldiphosphate synthase (FPPS) (references 5-12) resulting in decreasedlevels of protein prenylation in osteoclasts (references 13-15). Certainbisphosphonates have also been found to have anti-parasitic activity(references 16-25) and have been found to stimulate human γδ T cells(references 26-30); there is currently interest in their use as vaccinesfor a variety of B cell malignancies (reference 31).

Differences in substituents, however, can strongly influence thepharmacologic properties of such compounds (Green, 2001). Structuraldifferences may also be significant in the potential expansion oftherapies. For example, Bonefos (clodronate) is a bisphosphonateindicated for the treatment of tumor-induced osteolysis andhypercalcemia. It has been reported to increase survival and reduce therisk of bone metastasis in women with stage II/III breast cancer. Thisis noteworthy as approximately 70% of women who develop recurrence ofbreast cancer will experience bone metastasis, and breast cancer remainsthe leading cause of death among women aged 40 to 55 years.

For even second generation bisphosphonates, it is recognized that smallchanges of structure can lead to marked improvements in activity orfunction, for example in the inhibition of osteoclastic resorptionpotency (Widler et al., 2002). Therefore, there is great interest in thefurther development of alternative bisphosphonate compounds and theexploration of methods of use such as clinical applications.

SUMMARY OF THE INVENTION

The present invention surprisingly provides the first report of thesynthesis and testing of a series of pyridinium-1-yl and relatedbisphosphonates. Bisphosphonate compounds of the invention candemonstrate activity in one or more contexts, including a farnesyldiphosphate synthase (FPPS) assay, a D. discoideum growth inhibitionassay, a T cell activation assay, a bone resorption assay, the treatmentof infectious disease, the treatment of a bone resorption clinicaldisorder, an immunotherapeutic treatment, the treatment of cancer, andthe treatment of bone pain.

The invention broadly provides bisphosphonate compounds and relatedmethods of making and using. The invention specifically providescompounds with an N-linkage including pyridinium-1-yl, quinolinium-1-yl,and related bisphosphonate compounds.

The following abbreviations are applicable. FPPS, farnesyl diphosphatesynthase; pIC₅₀/pEC₅₀, negative log of IC₅₀ and EC₅₀, respectively,where IC₅₀ and EC₅₀ are the concentrations that produce half-maximalinhibition or activation, respectively; L. major, Leishmania major; D.discoideum, Dictyostelium discoideum; γδ T cells, gammadelta T cells.Compounds are optionally designated by a number or in some cases anumber preceded by a letter to help distinguish a compound designationfrom a cardinal number, e.g. C1 is compound 1.

The following definitions are applicable.

Alkyl groups include straight-chain, branched and cyclic alkyl groups.Alkyl groups include those having from 1 to 20 carbon atoms. Alkylgroups include small alkyl groups having 1 to 3 carbon atoms. Alkylgroups include medium length alkyl groups having from 4-10 carbon atoms.Alkyl groups include long alkyl groups having more than 10 carbon atoms,particularly those having 10-20 carbon atoms. Cyclic alkyl groupsinclude those having one or more rings. Cyclic alkyl groups includethose having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring andparticularly those having a 3-, 4-, 5-, 6-, or 7-member ring. The carbonrings in cyclic alkyl groups can also carry alkyl groups. Cyclic alkylgroups can include bicyclic and tricyclic alkyl groups. Alkyl groupsoptionally include substituted alkyl groups. Substituted alkyl groupsinclude among others those which are substituted with aryl groups, whichin turn can be optionally substituted. Specific alkyl groups includemethyl, ethyl, n-propyl, iso-propyl, cyclopropyl, n-butyl, s-butyl,t-butyl, cyclobutyl, n-pentyl, branched-pentyl, cyclopentyl, n-hexyl,branched hexyl, and cyclohexyl groups, all of which are optionallysubstituted.

Alkenyl groups include straight-chain, branched and cyclic alkenylgroups. Alkenyl groups include those having 1, 2 or more double bondsand those in which two or more of the double bonds are conjugated doublebonds. Alkenyl groups include those having from 2 to 20 carbon atoms.Alkenyl groups include small alkyl groups having 2 to 3 carbon atoms.Alkenyl groups include medium length alkenyl groups having from 4-10carbon atoms. Alkenyl groups include long alkenyl groups having morethan 10 carbon atoms, particularly those having 10-20 carbon atoms.Cyclic alkenyl groups include those having one or more rings. Cyclicalkenyl groups include those in which a double bond is in the ring or inan alkenyl group attached to a ring. Cyclic alkenyl groups include thosehaving a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring andparticularly those having a 3-, 4-, 5-, 6- or 7-member ring. The carbonrings in cyclic alkenyl groups can also carry alkyl groups. Cyclicalkenyl groups can include bicyclic and tricyclic alkyl groups. Alkenylgroups are optionally substituted. Substituted alkenyl groups includeamong others those which are substituted with alkyl or aryl groups,which groups in turn can be optionally substituted. Specific alkenylgroups include ethenyl, prop-1-enyl, prop-2-enyl, cycloprop-1-enyl,but-1-enyl, but-2-enyl, cyclobut-1-enyl, cyclobut-2-enyl, pent-1-enyl,pent-2-enyl, branched pentenyl, cyclopent-1-enyl, hex-1-enyl, branchedhexenyl, cyclohexenyl, all of which are optionally substituted.

Aryl groups include groups having one or more 5- or 6-member aromatic orheteroaromatic rings. Aryl groups can contain one or more fused aromaticrings. Heteroaromatic rings can include one or more N, O, or S atoms inthe ring. Heteroaromatic rings can include those with one, two or threeN, those with one or two O, and those with one or two S. Aryl groups areoptionally substituted. Substituted aryl groups include among othersthose which are substituted with alkyl or alkenyl groups, which groupsin turn can be optionally substituted. Specific aryl groups includephenyl groups, biphenyl groups, pyridinyl groups, and naphthyl groups,all of which are optionally substituted.

Arylalkyl groups are alkyl groups substituted with one or more arylgroups wherein the alkyl groups optionally carry additional substituentsand the aryl groups are optionally substituted. Specific alkylarylgroups are phenyl-substituted alkyl groups, e.g., phenylmethyl groups.

Alkylaryl groups are aryl groups substituted with one or more alkylgroups wherein the alkyl groups optionally carry additional substituentsand the aryl groups are optionally substituted. Specific alkylarylgroups are alkyl-substituted phenyl groups such as methylphenyl.

The rings that may be formed from two or more of R¹-R⁵ together can beoptionally substituted cycloalkyl groups, optionally substitutedcycloalkenyl groups or aromatic groups. The rings may contain 3, 4, 5,6, 7 or more carbons. The rings may be heteroaromatic in which one, twoor three carbons in the aromatic ring are replaced with N, O or S. Therings may be heteroalkyl or heteroalkenyl, in which one or more CH₂groups in the ring are replaced with O, N, NH, or S.

Optional substitution of any alkyl, alkenyl and aryl groups includessubstitution with one or more of the following substituents: halogens,—CN, —COOR, —OR, —COR, —OCOOR, —CON(R)₂, —OCON(R)₂, —N(R)₂, —NO₂, —SR,—SO₂R, —SO₂N(R)₂ or —SOR groups. Optional substitution of alkyl groupsincludes substitution with one or more alkenyl groups, aryl groups orboth, wherein the alkenyl groups or aryl groups are optionallysubstituted. Optional substitution of alkenyl groups includessubstitution with one or more alkyl groups, aryl groups, or both,wherein the alkyl groups or aryl groups are optionally substituted.Optional substitution of aryl groups includes substitution of the arylring with one or more alkyl groups, alkenyl groups, or both, wherein thealkyl groups or alkenyl groups are optionally substituted.

Optional substituents for alkyl, alkenyl and aryl groups include amongothers:

—COOR where R is a hydrogen or an alkyl group or an aryl group and morespecifically where R is methyl, ethyl, propyl, butyl, or phenyl groupsall of which are optionally substituted;

—COR where R is a hydrogen, or an alkyl group or an aryl groups and morespecifically where R is methyl, ethyl, propyl, butyl, or phenyl groupsall of which groups are optionally substituted;

—CON(R)₂ where each R, independently of each other R is a hydrogen or analkyl group or an aryl group and more specifically where R is methyl,ethyl, propyl, butyl, or phenyl groups all of which groups areoptionally substituted; R and R can form a ring which may contain one ormore double bonds;

—OCON(R)₂ where each R, independently of each other R, is a hydrogen oran alkyl group or an aryl group and more specifically where R is methyl,ethyl, propyl, butyl, or phenyl groups all of which groups areoptionally substituted; R and R can form a ring which may contain one ormore double bonds;

—N(R)₂ where each R, independently of each other R, is a hydrogen, or analkyl group, acyl group or an aryl group and more specifically where Ris methyl, ethyl, propyl, butyl, or phenyl or acetyl groups all of whichare optionally substituted; or R and R can form a ring which may containone or more double bonds.

—SR, —SO₂R, or —SOR where R is an alkyl group or an aryl groups and morespecifically where R is methyl, ethyl, propyl, butyl, phenyl groups allof which are optionally substituted; for —SR, R can be hydrogen;

—OCOOR where R is an alkyl group or an aryl groups;

—SO₂N(R)₂ where R is a hydrogen, an alkyl group, or an aryl group and Rand R can form a ring;

—OR where R═H, alkyl, aryl, or acyl; for example, R can be an acylyielding —OCOR* where R* is a hydrogen or an alkyl group or an arylgroup and more specifically where R* is methyl, ethyl, propyl, butyl, orphenyl groups all of which groups are optionally substituted;

Specific substituted alkyl groups include haloalkyl groups, particularlytrihalomethyl groups and specifically trifluoromethyl groups. Specificsubstituted aryl groups include mono-, di-, tri, tetra- andpentahalo-substituted phenyl groups; mono-, di-, tri-, tetra-, penta-,hexa-, and hepta-halo-substituted naphthalene groups; 3- or4-halo-substituted phenyl groups, 3- or 4-alkyl-substituted phenylgroups, 3- or 4-alkoxy-substituted phenyl groups, 3- or4-RCO-substituted phenyl, 5- or 6-halo-substituted naphthalene groups.More specifically, substituted aryl groups include acetylphenyl groups,particularly 4-acetylphenyl groups; fluorophenyl groups, particularly3-fluorophenyl and 4-fluorophenyl groups; chlorophenyl groups,particularly 3-chlorophenyl and 4-chlorophenyl groups; methylphenylgroups, particularly 4-methylphenyl groups, and methoxyphenyl groups,particularly 4-methoxyphenyl groups.

Pharmaceutically acceptable salts comprise pharmaceutically-acceptableanions and/or cations. Pharmaceutically-acceptable cations include amongothers, alkali metal cations (e.g., Li⁺, Na⁺, K⁺), alkaline earth metalcations (e.g., Ca²⁺, Mg²⁺), non-toxic heavy metal cations and ammonium(NH₄ ⁺) and substituted ammonium (N(R′)₄ ⁺, where R′ is hydrogen, alkyl,or substituted alkyl, i.e., including, methyl, ethyl, or hydroxyethyl,specifically, trimethyl ammonium, triethyl ammonium, and triethanolammonium cations). Pharmaceutically-acceptable anions include amongother halides (e.g., Cl⁻, Br⁻), sulfate, acetates (e.g., acetate,trifluoroacetate), ascorbates, aspartates, benzoates, citrates, andlactate.

Compounds of the invention can have prodrug forms. Prodrugs of thecompounds of the invention are useful in the methods of this invention.Any compound that will be converted in vivo to provide a biologically,pharmaceutically or therapeutically active form of a compound of theinvention is a prodrug. Various examples and forms of prodrugs are wellknown in the art. Examples of prodrugs are found, inter alia, in Designof Prodrugs, edited by H. Bundgaard, (Elsevier, 1985), Methods inEnzymology, Vol. 42, at pp. 309-396, edited by K. Widder, et. al.(Academic Press, 1985); A Textbook of Drug Design and Development,edited by Krosgaard-Larsen and H. Bundgaard, Chapter 5, “Design andApplication of Prodrugs,” by H. Bundgaard, at pp. 113-191, 1991); H.Bundgaard, Advanced Drug Delivery Reviews, Vol. 8, p. 1-38 (1992); H.Bundgaard, et al., Journal of Pharmaceutical Sciences, Vol. 77, p. 285(1988); and Nogrady (1985) Medicinal Chemistry A Biochemical Approach,Oxford University Press, New York, pages 388-392).

The invention provides compounds having the formula CA1:

(see also FIG. 9)

or a pharmaceutically acceptable salt, ester or hydrate thereof;wherein:

X is H, —OH, or a halogen;

n is 1, 2, or 3;

R¹-R⁵, independently of one another and other R groups, are selectedfrom the group consisting of a hydrogen, a halogen, a —CN, —OR, —COOR,—OCOOR, —COR, —CON(R)₂, —OCON(R)₂, —N(R)₂, —NO₂, —SR, —SO₂R, —SO₂N(R)₂or —SOR group, an optionally substituted alkyl group, an optionallysubstituted alkenyl group, and an optionally substituted aryl group,where each R, independent of any other R in any listed group, isselected from H, an optionally substituted alkyl group, an optionallysubstituted aryl group, and an optionally substituted acyl group;

two or more of R¹-R⁵ can together form one or more rings which maycontain one or more double bonds or which may be aromatic;

R⁶ and R⁷, independently of each other and other R⁶ and R⁷ in thecompound, are selected from the group consisting of a hydrogen, ahalogen, a —N(R)₂, or —SR group, an optionally substituted alkyl group,an optionally substituted alkenyl group, an optionally substitutedalkoxy group, and an optionally substituted aryl group, where each R,independent of any other R in any listed group, is selected from H, anoptionally substituted alkyl group and an optionally substituted arylgroup; and

wherein R⁶ and R⁷ can together form a ring which may contain one or moredouble bonds.

In specific embodiments, the invention relates to compounds having theabove formula where X is OH.

In other specific embodiments, the invention relates to compounds havingthe above formula where X is H.

In other specific embodiments, compounds of the invention are those offormula CA1, with the exception of the compound of formula CA1 where Xis H, n is 1 and all of R¹-R⁷ are hydrogens.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein n is 1.

In other specific embodiments, the invention relates to compounds havingthe above formula where X is OH and n is 1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein one or both of R⁶ and R⁷ are hydrogens.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein both of R⁶ and R⁷ are hydrogens.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein both of R⁶ and R⁷ are hydrogens and n is 1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein both of R⁶ and R⁷ are hydrogens, n is 1 and Xis OH.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein both of R⁶ and R⁷ are hydrogens, n is 1 and Xis H.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹-R⁵ are all hydrogens.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹-R⁵ are all hydrogens, X is OH and n is 1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹-R⁵ are all hydrogens, X is H and n is 1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogen, X is OH and n is1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogen, X is H and n is1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogen and one or more ofR², R³ or R⁴ is a halogen.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, one or more ofR², R³ or R⁴ is a halogen, X is OH and n is 1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, one or more ofR², R³ or R⁴ is a halogen, X is H and n is 1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, and one or moreof R², R³ or R⁴ is an optionally substituted alkyl group, particularly asmall alkyl group and more particularly a methyl group.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, one or more ofR², R³ or R⁴ is an optionally substituted alkyl group, particularly asmall alkyl group, X is OH and n is 1. Specific compounds of thisinvention are those as above in which R¹ and R⁵ are both hydrogens, oneor more of R², R³ or R⁴ is a methyl group, X is OH and n is 1. Specificcompounds of this invention are those as above in which R¹ and R⁵ areboth hydrogens, one or more of R², R³ or R⁴ is a trifluoromethyl group,X is OH and n is 1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, one or more ofR², R³ or R⁴ is an optionally substituted alkyl group, particularly asmall alkyl group, X is H and n is 1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, and one or moreof R², R³ or R⁴ is an optionally substituted alkoxy group. A specificalkoxy group is a methoxy group. Specific compounds of this inventionare those as in the formula above in which R² or R³ is a methoxy group.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, and one or moreof R², R³ or R⁴ is an optionally substituted alkoxy group, X is OH and nis 1. Specific compounds of the invention are those as in the formulaabove wherein R¹ and R⁵ are both hydrogens, R² or R³ is a methoxy group,X is OH and n is 1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, and one or moreof R², R³ or R⁴ is an optionally substituted alkoxy group, X is H and nis 1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, and one or moreof R², R³ or R⁴ is an optionally substituted phenyl group.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, one or more ofR², R³ or R⁴ is an optionally substituted phenyl group, X is OH and n is1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, one or more ofR², R³ or R⁴ is an optionally substituted phenyl group, X is H and n is1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, and one or moreof R², R³ or R⁴ is an alkyl-substituted phenyl group. Specific alkylgroups are methyl, ethyl and n-propyl groups. Specific compounds of thisinvention are those as above in which R² or R³ is a 3- or 4-alkylphenylgroup. Specific compounds of this invention are those as above in whichR² or R³ is a 3- or 4-methylphenyl group. Specific compounds of thisinvention are those as above in which R² or R³ is a 3- or 4-ethylphenylgroup. Specific compounds of this invention are those as above in whichR² or R³ is a 3- or 4-n-butylphenyl group.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, one or more ofR², R³ or R⁴ is an alkyl-substituted phenyl group, X is OH and n is 1.Specific alkyl groups are methyl, ethyl and n-propyl groups. Specificcompounds of this invention are those as above in which R² or R³ is a 3-or 4-methylphenyl group, X is OH and n is 1. Specific compounds of thisinvention are those as above in which R² or R³ is a 3- or 4-ethylphenylgroup, X is OH and n is 1. Specific compounds of this invention arethose as above in which R² or R³ is a 3- or 4-n-propylphenyl group, X isOH and n is 1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, one or more ofR², R³ or R⁴ is an alkyl-substituted phenyl group, X is H and n is 1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, and one or moreof R², R³ or R⁴ is a halo-substituted phenyl group. Specific halogensare fluorine, chlorine and bromine. Specific compounds of this inventionare those as above in which R² or R³ is a 3- or 4-halophenyl group or a3-, 4-dihalophenyl group. Specific compounds of this invention are thoseas above in which R² or R³ is a 3- or 4-fluorophenyl group. Specificcompounds of this invention are those as above in which R² or R³ is a 3-or 4-chlorophenyl group. Specific compounds of this invention are thoseas above in which R² or R³ is a 3- or 4-bromophenyl group. Specificcompounds of this invention are those as above in which R² or R³ is a3-bromo-4-fluorophenyl group or a 3-chloro-4-fluorophenyl group.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, one or more ofR², R³ or R⁴ is a halo-substituted phenyl group, X is OH and n is 1.Specific halogens are fluorine, chlorine, and bromine. Specificcompounds of this invention are those as above in which R² or R³ is a 3-or 4-fluorophenyl group, X is OH and n is 1. Specific compounds of thisinvention are those as above in which R² or R³ is a 3- or 4-chlorophenylgroup, X is OH and n is 1. Specific compounds of this invention arethose as above in which R² or R³ is a 3- or 4-bromophenyl group, X is OHand n is 1. Specific compounds of this invention are those as above inwhich R² or R³ is a 3-bromo-4-fluorophenyl group or a3-chloro-4-fluorophenyl group, X is OH and n is 1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, one or more ofR², R³ or R⁴ is a halo-substituted phenyl group, X is H and n is 1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, and one or moreof R², R³ or R⁴ is a hydroxy-substituted phenyl group which may be inthe form of a phenoxy anion or salt thereof. Specific compounds of thisinvention are those as above in which R² or R³ is a 3- or4-hydroxyphenyl group. Specific compounds of this invention are those asabove in which R² or R³ is a 3- or 4-hydroxyphenyl group. Specificcompounds of this invention are those in which R² or R³ is a 3- or4-oxyphenyl anion or a salt thereof. Salts of the oxyphenyl anioninclude Na⁺, K⁺, and other pharmaceutically acceptable salts containingpharmaceutically acceptable cations.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, one or more ofR², R³ or R⁴ is a hydroxy-substituted phenyl group, which may be in theform of a phenoxy anion or salt thereof, X is OH and n is 1. Specificcompounds of this invention are those as above in which R² or R³ is a 3-or 4-hydroxyphenyl group, X is OH and n is 1. Specific compounds of thisinvention are those in which R² or R³ is a 3- or 4-oxyphenyl anion or asalt thereof, X is OH, and n is 1. Salts of the oxyphenyl anion includeNa⁺, K⁺, and other pharmaceutically acceptable salts containingpharmaceutically acceptable cations.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, and one or moreof R², R³ or R⁴ is an alkoxy-substituted phenyl group. A specific alkoxygroup is a methoxy group. Specific compounds of this invention are thoseas above in which R² or R³ is a 3- or 4-methoxy phenyl group.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, one or more ofR², R³ or R⁴ is an alkoxy substituted phenyl group, X is OH and n is 1.A specific alkoxy group is a methoxy group. Specific compounds of thisinvention are those as above in which R² or R³ is a 3- or 4-methoxyphenyl group, X is OH and n is 1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, and one or moreof R², R³ or R⁴ is an optionally substituted arylalkyl group. A specificarylalkyl group is a phenylmethyl group, particularly the compound asabove wherein R² or R³ is a phenylmethyl group.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, one or more ofR², R³ or R⁴ is an optionally substituted arylalkyl group, X is OH and nis 1. A specific compound of this invention is one in which R¹ and R⁵are both hydrogens, R² or R³ is a phenylmethyl group, X is OH and n is1.

In other specific embodiments, the invention relates to compounds havingthe above formula wherein R¹ and R⁵ are both hydrogens, one or more ofR², R³ or R⁴ is an optionally substituted arylalkyl group, X is H and nis 1.

In a particular embodiment of CA1, X is OH, n=1, and R¹-R⁷ are H. In aparticular embodiment of CA1, X is a halogen. In a more particularembodiment, the halogen is selected from the group consisting of Cl orF. In an embodiment of CA1, X is Cl. In an embodiment of CA1, X is F. Inan embodiment of CA1, X is not H.

In other specific embodiments, the invention includes compounds offormula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH orH and R² is selected from the group consisting of H, optionallysubstituted alkyl groups, optionally substituted alkoxy groups andoptionally substituted phenyl groups. Of particular interest are thosecompounds in which the optional substitution is one or more halogens,including one or more fluorines or chlorines.

In other specific embodiments, the invention includes compounds offormula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH andR² is selected from the group consisting of H, optionally substitutedalkyl groups, optionally substituted alkoxy groups and optionallysubstituted phenyl groups. Of particular interest are those compounds inwhich the optional substitution is one or more halogens, including oneor more fluorines or chlorines.

In other specific embodiments, the invention includes compounds offormula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH orH and R² is selected from the group consisting of optionally substitutedalkyl groups, optionally substituted alkoxy groups and optionallysubstituted phenyl groups. Of particular interest are those compounds inwhich the optional substitution is one or more halogens, including oneor more fluorines or chlorines.

In other specific embodiments, the invention includes compounds offormula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH andR² is selected from the group consisting of optionally substituted alkylgroups, optionally substituted alkoxy groups and optionally substitutedphenyl groups. Of particular interest are those compounds in which theoptional substitution is one or more halogens, including one or morefluorines or chlorines.

In other specific embodiments, the invention includes compounds offormula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH orH and R² is selected from the group consisting of H, alkyl groups,alkoxy groups and a phenyl group.

In other specific embodiments, the invention includes compounds offormula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH andR² is selected from the group consisting of H, alkyl groups, alkoxygroups and a phenyl group.

In other specific embodiments, the invention includes compounds offormula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH orH and R² is selected from the group consisting of H, a methyl group, anethyl group, propyl groups, butyl groups, a methoxy group, an ethoxygroup, propyloxy groups, butyloxy groups and a phenyl group.

In other specific embodiments, the invention includes compounds offormula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH andR² is selected from the group consisting of H, a methyl group, an ethylgroup, propyl groups, butyl groups, a methoxy group, an ethoxy group,propyloxy groups, butyloxy groups and a phenyl group.

In other specific embodiments, the invention includes compounds offormula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH orH and R² is selected from the group consisting of H, a methyl group, amethoxy group, and a phenyl group.

In other specific embodiments, the invention includes compounds offormula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH andR² is selected from the group consisting of H, a methyl group, a methoxygroup, and a phenyl group.

In a specific embodiment, compounds 278, 297, 300 and 446; andpharmaceutically acceptable salts, and esters thereof; are useful fortreatment of a bone resorption clinical disorder.

In a specific embodiment, compounds 278, 297, 300, 444, 445 and 446; andpharmaceutically acceptable salts, and esters thereof; are useful intreatment of protozoan diseases, useful for treatment of a boneresorption clinical disorder, and for immunotherapy.

In a specific embodiment, compounds, the des-hydroxy (where X is H)analogs of compounds 278, 297, 300, 444, 445 and 446; andpharmaceutically acceptable salts, and esters thereof; are useful in thetreatment of a bone resorption clinical disorder.

Compounds of this invention and compounds useful in the methods of thisinvention include those of the above formulas andpharmaceutically-acceptable salts and esters of those compounds. Saltsinclude any salts derived from the acids of the formulas herein whichacceptable for use in human or veterinary applications. The term estersrefers to hydrolyzable esters of diphosphonate compounds of the formulasherein. Salts and esters of the compounds of the formulas herein arethose which have the same therapeutic or pharmaceutical (human orveterinary) properties as the diphosphonate compounds of the formulasherein. Various combinations of salts are possible, with eachphosphonate carrying a 2-, 1- or neutral charge. In principle there aremultiple charge states possible, for example 9 charge states, forcertain bisphosphonates of this invention.

In an embodiment, the invention provides a compound selected from thegroup consisting of 278, 297, 300, 335, 344, 359, 364, 398, 443-447,449-452, 455-457, 459-462,470-481, 483-485, ZZ1, 502, 511, 513, 520,521, 523-526, 529-534, 542, 556, 577-579, 582, 583, 586, 588, 590, 591,595, 597-605, 607, 610, 612, and 613; and for each respective saidcompound, a pharmaceutically acceptable salt or ester thereof.

In an embodiment, the invention provides a therapeutic compositioncomprising one or more compounds selected from the group consisting of278, 297, 300, 335, 344, 359, 364, 398, 443-447, 449-452, 455-457,459-462, 470-481, 483-485, ZZ1; and for each numbered compound apharmaceutically acceptable salt or ester thereof; wherein the compoundsare present in the composition in an amount or in a combined amounteffective for obtaining the desired therapeutic benefit. The therapeuticcompositions of this invention optionally further comprise apharmaceutically acceptable carrier as known in the art.

In a specific embodiment, the invention includes compounds of the aboveformula CA1 where n=1, R¹ and R³-R⁷ are hydrogens, X═OH, and R²═H,optionally substituted alkyl, optionally substituted alkoxy, andoptionally substituted phenyl. In a more specific embodiment, theinvention includes compounds where n=1, R¹ and R³-R⁷═H, X═OH, and R²═H,alkyl, alkoxy, and phenyl. In a further specific embodiment, theinvention includes compounds where n=1, R¹ and R³-R⁷═H, X═OH, and R²═H,methyl, ethyl, propyl, butyl, methoxy, ethoxy, propoxy, butoxy, orphenyl.

In an embodiment, the invention provides various methods relating to thetreatment of clinical disease. In an embodiment, the invention providesa method of treating a bone resorption disorder comprising administeringto a patient in need a composition comprising a compound of theinvention.

In an embodiment, the invention provides a method of treating a cancerdisorder comprising administering to a patient in need a compositioncomprising a compound of the invention. In a specific embodiment, thecancer is breast cancer. In a specific embodiment, the breast cancerinvolves an actual or potential bone metastatic condition. In a specificembodiment, the invention provides a method of treating myeloma,lymphoma, prostate cancer, an epidermoid cancer, or orthotopic tumors.

In an embodiment, the invention provides compounds and methods for usein a combination therapy in the treatment of cancer. In a specificembodiment, a combination therapy utilizes a bisphosphonate compound ofthe invention and a different chemotherapeutic agent which canoptionally be a distinct other bisphosphonate compound. In a particularembodiment the different chemotherapeutic agent is alendronate,zoledronate, risedronate, pamidronate, fas ligand (FasL), mevastatin,dexamethasone, paclitaxel, epirubicin, docetaxel, imatinib mesylate,tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL),uracil-tegafur, gemcitabine, melphalan, doxorubicin, vincristine, orR115777 farnesyl transferase inhibitor (FTI) (Zarnestra®). In aparticular embodiment, the combination of the bisphosphonate compound ofthe invention and the different chemotherapeutic agent has a synergisticeffect. In another particular embodiment the combination has an additiveeffect.

In an embodiment, the invention provides a method of treating aninfectious disease comprising administering to a patient in need acomposition comprising a compound of the invention. In a specificembodiment, the infectious disease relates to an agent selected from thegroup consisting of: a virus, a bacterium, a fungus, and a protozoanparasite. In a specific embodiment, the virus is a retrovirus. In a morespecific embodiment, the retrovirus is human immunodeficiency virus(HIV). In an embodiment, the protozoan parasite is Leishmania major. Inan embodiment, the protozoan parasite is selected from the groupconsisting of: Leishmania, Toxoplasma, Cryptosporidium, Plasmodium, andTrypanosoma. In an embodiment, the infectious disease is selected fromthe group consisting of leishmaniasis, toxoplasmosis, cryptosporidiosis,sleeping sickness, and malaria.

In an embodiment, the invention provides a method of immunotherapycomprising administering to a patient in need a composition comprising acompound of the invention. In a specific embodiment, the methodstimulates T cells in the patient. In a more specific embodiment, themethod stimulates gamma delta T cells.

In an embodiment, the invention provides a method of screening abisphosphonate test compound for a potential therapeutic activity,comprising: providing said bisphosphonate test compound, measuring aperformance attribute of said test compound in at least three assaysselected from the group consisting of: a Leishmania major farnesyldiphosphate synthase (FPPS) assay, a Dictyostelium discoideum assay, a Tcell activation assay, and a bone resorption assay, analyzing saidperformance attribute; and selecting said bisphosphonate test compoundbased on said attribute; thereby screening said bisphosphonate testcompound. In a specific embodiment, the method further comprisesproviding a reference compound and comparing a performance attribute ofsaid reference compound with said performance attribute of said testcompound.

In an embodiment, the invention provides a method of synthesizing abisphosphonate compound of the invention, for example formula CA1,comprising: syntheses as shown and described herein, e.g. in schemes,FIG. 2, etc.; and as further would be understood in the art.

In an embodiment, the invention provides a method of treating bone paincomprising administering to a patient in need a compound of theinvention. In a particular embodiment, the treatment of bone pain is inthe context of a bone disease. In a particular embodiment, the treatmentof bone pain is in the context of a patient with a metastatic cancer. Ina particular embodiment, the metastatic cancer has spread to a bonelocation or originated in a bone location. For example, the treatment ofbone pain can be achieved in a breast cancer patient wherein ametastatic breast cancer can or has spread to a bone location.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates compounds C1-C6, including pamidronate (Aredia®) C1;alendronate (Fosamax®) C2; risedronate (Actonel®) C3; zoledronate(Zometa®) C4; C5; and C6 shown in their zwitterionic forms.

FIG. 2 illustrates synthetic routes in the preparation of compounds.

FIG. 3 illustrates structures of compounds.

FIG. 4 illustrates structures of selected compounds.

FIG. 5 illustrates structures of selected compounds.

FIG. 6 illustrates cellular pathways of isoprenoid biosynthesis.

FIG. 7 illustrates the structures of pyridinium-1-yl bisphosphonates,compounds C5 and C7-C18.

FIG. 8 illustrates correlations: A, between FPPS inhibition and D.discoideum growth inhibition; and B, between FPPS inhibition and γδ Tcell activation (as determined by TNF-α release). The R² and p valuesare R²=0.65 and p<0.0001 in A and R²=0.68 and p<0.0001 in B.

FIG. 9 illustrates a structure of a bisphosphonate compound.

DETAILED DESCRIPTION OF THE INVENTION

The invention may be further understood by the following non-limitingexamples.

EXAMPLE 1 Bisphosphonate Compounds

We report the design, synthesis and testing of a series of novelbisphosphonates. The most potent molecules have high activity and canrepresent useful compositions for a variety of applications such as inbone resorption disorders, parasitic diseases, immunomodulation, andcancer.

Our efforts led to the prediction of the importance of the presence of apositive charge at a relatively localized position in the bisphosphonateside chain. This can be related to the position of the positive chargeexpected in the pyridinium and imidazolium forms of compounds C3 and C4,shown above.

We explored the possibility that the pyridinium-1-yl species, compoundC5, might have useful activity. We synthesized C5 and a series ofderivatives (compounds C7-C18, FIG. 7), using the following generalscheme, Scheme 1.

In the first step, we used (where necessary) coupling reactions ofarylmetallic compounds with bromopyridines, catalyzed by Pd(PPh₃)₄ (ref.32) or NiCl₂(PPh₃)₂ (ref. 33) to produce substituted pyridines. Thesubstituted pyridines were then alkylated by using bromoacetic acid(ref. 34) and the resulting pyridinium-1-yl acetic acids were convertedto the corresponding bisphosphonates by using H₃PO₃/POCl₃ (ref. 35).

We then investigated the activity of each compound in inhibiting theFPPS from L. major, in Dictyostelium discoideum growth inhibition and inγδ T cell activation. We used the FPPS from L. major since this enzymeis the putative bisphosphonate target in several trypanosomatid species.We used D. discoideum to test for cell growth inhibition, since thisorganism has been useful in the context of the development of boneresorption drugs (ref. 36). To determine the stimulatory activity ofcompounds C5 and C7-C18 for Vγ2Vδ2 T cells, we used the TNF-α releaseassay (ref. 30).

In the L. major FPPS inhibition assay, C5 was found to have a Ki of 18nM (Table 6) and was thus slightly less active than the most potentcommercially available bisphosphonates, zoledronate (4, Ki=11 nM in thisassay) and risedronate (3, Ki=17 nM in this assay) (Table 6). In orderto try to enhance activity, we next investigated the desirability ofplacing substituents at the meta position. We thus prepared compoundsC7-C9 (FIG. 7) and tested them in the FPPS assay. The meta-methyl analogof C5 (C7) was not as active as C5 (Ki=38 nM versus 18 nM), butsubstitution with a meta-phenyl group, giving C8, resulted in a verypotent species, having a Ki=9 nM, slightly more active than zoledronate,4 (Ki=11 nM). The para-phenoxy derivative of C5 (C9) was found to beless active (Ki=75 nM), possibly due to unfavorable electrostaticinteractions of the OH group in the FPPS active site.

TABLE 1 Activities of bisphosphonates as L. major FPPS inhibitors, D.discoideum cell growth inhibitors and gammadelta T cell simulators.*Com- L. major D. discoideum gammadelta T cell pound FPPS Ki IC₅₀stimulation, Compound Alias (nM) (μM) EC₅₀ (μM)  1 (pamid- 190 167 940ronate)^(a,b)  2 (alen- 95 32 52 dronate)^(a,b)  3 (rised- 17 2.8 6.2ronate)^(a,b)  4 (zole- 11 1.9 7.3 dronate)^(a,b)  5 278 18 2.1 5.1  6(incad- 23 1.6 15 ronate)^(a,b)  7 297 38 1.5 4.6  8 300 9 2.8 3.7  9359 75 2.9 24 10 335 160 8.5 430 11 344 70 9.9 140 12 364 950 2.3 >100013 398 80 20 41 14 447 380 72 53 15 444 20 5.6 5.1 16 445 20 2.9 5.2 17446 30 6 4.6 18 443 110 12 230 ^(a) L. major FPPS inhibition data fromRef. 17 ^(b)Gammadelta T cell stimulation data from Ref. 30 *Note thatcompound designations here are referred to by numbers and may optionallybe designated with a preceding “C” yielding, e.g., C1, C2, etc.

Since the phenylpyridinium species, C8, displayed good activity, we nextsynthesized C10 and C11. Both of these compounds contain a methylenelinker between the two aromatic groups and it seemed possible that theymight better mimic the putative geranyl diphosphate reactiveintermediate (C19):

but each of these compounds was approximately ten-fold less active thanC8, with Ki values in the 70-160 nM range, Table 1. We also prepared thebiphenylpyridinium compound, C12, since the added hydrophobicity of C8appeared encouraging, but C12 proved to be relatively inactive, having aKi of 950 nM. The isoquinoline and quinoline species, C13 and C14, hadmodest activity (80 and 380 nM, respectively, for C13 and C14), but themeta-ethyl (C15), butyl (C16), methoxy (C17) and para-benzyl (C18)pyridinium species were generally more active (20, 20, 30 and 110 nM,respectively), although they were less active than compounds C3-C6, C8.See Table 1.

We next investigated D. discoideum growth inhibition by compounds 1-18(Table 1; alternatively referred to as C1-C18). The most active compoundfound was the meta-methylpyridinium compound, C7, which had an IC₅₀ of1.5 μM, followed by incadronate (C6, IC₅₀=1.6 μM) and zoledronate (C4,IC₅₀=1.9 μM). The unsubstituted pyridinium bisphosphonate, C5, wasslightly more active (IC₅₀=2.1 μM) than was risedronate (C3, IC₅₀=2.8μM). As with the FPPS inhibition results, the benzylpyridiniumbisphosphonates (C10, C11, C18) were less active than the pyridinium andphenylpyridinium species (C5, C7-9). Surprisingly, C12 showed highactivity, due perhaps to the possibility of an additional target in D.discoideum or the possibility of structural differences between L. majorand D. discoideum FPPS enzymes. With the exception of C12, the activityresults for the 17 bisphosphonates are highly correlated (R²=0.65,p<0.0001), as shown in FIG. 8A.

Next, we investigated the ability of C5 and compounds C7-C18 tostimulate gammadelta T cells, using the TNF-α release assay (ref. 30).The most active compound was found to be C8 (EC₅₀=3.7 μM), followed byC7 and C17 (EC₅₀=4.6 μM), with these compounds having more activity thanrisedronate (C3, EC₅₀=6.2 μM) (ref. 30) or zoledronate (C4, EC₅₀=7.3 μM)(ref. 30) in this TNF-α release assay. Addition of the para hydroxylgroup (8→9) again reduced activity (Table 1), and again all threemethylene bridged compounds (C10, C11, C18) had poor activity. Theactivity results for FPPS inhibition were found to be highly correlatedwith gammadelta T cell TNF-α release results (R²=0.68, p<0.0001), asshown in FIG. 8B, suggesting the likely importance of FPPS inhibition ingammadelta T cell activation (refs. 29, 30).

We also explored the idea that electron withdrawing substituents on thering could improve activity in bone resorption. To test this, compound461 was prepared and tested, and indeed this species was found to bevery potent in bone resorption. See Table 4.

Without wishing to be bound by a particular theory, these results mayconfirm the importance of a positive charge at the N−1 position. Thissuggests that the further development of this class of compounds cancontinue to be of interest in the context of the chemotherapy ofinfectious diseases, bone resorption, cancer, bone pain, and inimmunotherapy.

EXAMPLE 2 Structures of Bisphosphonate Compounds

Particular bisphosphonate compounds were synthesized. See Table 2 andthe following figures: FIG. 3, FIG. 4, and FIG. 5. Note that in Table 2,the first column with the heading “Item” is not intended to refer to acompound designation, whereas the second column does refer to compounddesignations.

TABLE 2 Summary list of compounds with corresponding structuresillustrated in FIGS. 3-5. Item Compound Designation 1 278 2 297 3 300 4335 5 344 6 359 7 364 8 398 9 443 10 444 11 445 12 446 13 447 14 449 15450 16 451 17 452 18 455 19 456 20 457 21 459 22 460 23 461 24 462 25470 26 471 27 472 28 473 29 474 30 475 31 476 32 477 33 478 34 479 35480 36 481 37 483 38 484 39 485 40 ZZ1 41 502 42 511 43 513 44 520 45521 46 523 47 524 48 525 49 526 50 529 51 530 52 531 53 532 54 533 55534 56 542 57 556 58 577 59 578 60 579 61 582 62 583 63 586 64 588 65590 66 591 67 595 68 597 69 598 70 599 71 600 72 601 73 602 74 603 75604 76 605 77 607 78 610 79 612 80 613

EXAMPLE 3 Activity of Bisphosphonate Compounds in T Cell Stimulation andApplications in Immunotherapy

Additional compounds were tested for the ability to stimulate gammadeltaT cells. Results are shown in Table 3.

TABLE 3 gammadelta T cell stimulation results for selectedbisphosphonate compounds. Compound EC₅₀ (μM) 2 4.7 2 3.5 278 4.8 297 4.9300 2.1 335 63.4 398 40.6 442 27.5 443 186.6 444 2.8 445 2.5 461 2.7 4703.3 472 2.1 473 20.1 474 2.4 475 2.8 476 2.0 477 1.8 480 68.7 481 83.4482 12.5 483 2.9 484 4.6

EXAMPLE 4 Exploration of Activity of Bisphosphonate Compounds andStrategic Design of Compounds

We explored the hypothesis of whether analogs of pyrophosphate couldblock pyrophosphatase enzymes and/or inhibit cellular growth orfunction. We used certain bisphosphonate compounds that are currentlyapplied in bone resorption therapy. The compounds included pamidronate(Aredia®, Novartis), alendronate (Fosamax®, Merck), and risedronate(Actonel®, Procter & Gamble). The compounds did not appear necessarilyto inhibit pyrophosphatases but did inhibit parasite cell growth.Without wishing to be bound by a particular theory, the compoundsappeared to act as inhibitors of isoprenoid biosynthesis (FIG. 6).

The FPP synthase inhibitor pamidronate was observed to be effective intreating cutaneous leishmaniasis in mice. The average lesion size intreated mice was reduced during a time period of several weeks and in adose-dependent manner relative to treated mice.

In an embodiment, a compound of the invention inhibitsdeoxyxylulose-5-phosphate reductoisomerase (DXR), an enzyme involved inisoprenoid biosynthesis. In a particular embodiment, a compound is ableto affect Plasmodium in vitro or in vivo.

In an embodiment of the invention, a compound inhibits the mevalonatepathway. In an embodiment of the invention, a compound interacts withIPP isomerase (next to FPP synthase in the isoprene biosynthesispathway) and activates gammadelta T cells.

EXAMPLE 5 Activity of Bisphosphonate Compounds in Bone Resorption

Compounds are tested in a bone resorption assay: ⁴⁵Ca²⁺ release from17-day old fetal mouse metatarsals (ref. 37). Results of IC₅₀ values fortest compounds are observed and optionally compared to those forreference compounds such as risedronate (C3), alendronate (C2), andpamidronate (C1) and/or other bisphosphonate compounds known in the art.In a preferred embodiment, bisphosphonates of the invention such as thepyridinium-1-yl bisphosphonates are comparable to or more active in thebone resorption assay or in treatment of a bone resorption clinicaldisorder than one or more other reference bisphosphonates.

EXAMPLE 6 Application of Bisphosphonate Compounds in the Treatment ofCancer

Compounds are tested for efficacy in reducing the occurrence, severity,or course of bone metastases in stage II/III breast cancer patients. Acompound of the invention is found effective and administered to apatient in need of treatment. Treatment with a compound of the inventionis effective in reducing the risk of bone metastasis and/or increasingthe likelihood of survival, optionally in relation to treatment with aplacebo. A compound is effective in enhancing a survival outcome inpatients with more advanced disease. A compound administered to a cancerpatient can simultaneously provide a benefit in the treatment ofosteolysis and/or hypercalcemia while assisting in the prevention ofbone metastasis and significantly increasing overall survival in breastcancer patients.

Compositions of the invention are applied in the treatment of skinmetastases and mediastinal lymphomas. See Wilhelm et al., 2003.

Compositions of the invention are useful in the treatment of cancerssuch as lymphoma and myeloma and/or other forms of cancer. See Green JR, 2004, The Oncologist 9(supp 4):3-13; Forsea A-M et al., 2004, BritishJournal of Cancer 91:803-810.

Compositions of the invention are used in a combination therapy in thetreatment of cancer. In a specific embodiment, a combination therapyutilizes a bisphosphonate compound of the invention and a differentchemotherapeutic agent which can optionally be a distinct otherbisphosphonate compound. See Caraglia M et al., 2004, Oncogene23:6900-6913. See Salomo M et al., 2003, British Journal of Haematology122:202-210.

EXAMPLE 7 Application of Bisphosphonate Compounds in the Treatment ofHIV Infection and AIDS

Many HIV drugs are suboptimally effective in partial relation tomutations of HIV-1 reverse transcriptase that confer resistance to adrug. For example, the effectiveness of azidothymidine (AZT; zidovudine,Retrovir), is believed to be so diminished. Bisphosphonate compounds ofthe invention are used in conjunction with AZT to provide an improvedcomposition and therapy. Without wishing to be bound by a particulartheory, a bisphosphonate compound inhibits AZT excision caused by ATP orPPi; the inhibition results in increased AZT activity in enzyme andcellular assays. A reversion of resistance phenotype is achieved byrendering an HIV-1 strain more sensitive to AZT activity.

EXAMPLE 8 Additional Bisphosphonate Compounds

Further bisphosphonate compounds were synthesized. The structures areindicated in FIG. 9, FIG. 10, and FIG. 11. As noted in other Examplesherein, the functional activity of certain of these compounds wasassessed.

EXAMPLE 9 Bisphosphonate Compounds have Activity in Several ContextsRelating to Bone Resorption and Immune Regulation

We have obtained data for selected bisphosphonate compounds in thecontexts of D. discoideum assays, human FPPS assays, gammadelta T cellstimulation assays, and bone resorption assays. Several compounds showedsubstantial activity in one or more functional assays. Some toxicitytesting was also performed. See Table 4 (in some instances, the samedata depicted elsewhere herein, e.g., in Table 1, may be presented againto facilitate comparative analysis).

TABLE 4 (3) (5) (1) gammadelta (4) Toxicity IC₅₀ D. (2) T cell Bone(ug/mL - human discoideum human FPPS stimulation resorptionnasopharyngeal Compound IC₅₀ (uM) IC₅₀ (uM) EC₅₀ IC₅₀ (uM) carcinomacells) 278 2.6 1.6 5.1 0.67 300 297 2.8 NT 4.6 0.22 5.3 300 2.8 1.7 3.70.41 5.4 335 11 NT 430 NT NT 344 8.5 17 140 NT NT 359 2.9 NT 24 NT NT364 2.3 37 >1000 NT NT 398 20 NT 41 NT NT 443 12 NT 230 NT NT 444 5.6 NT5.1 NT NT 445 3 NT 5.2 NT NT 446 6 NT 4.6 0.37 NT 447 72 NT 53 NT NT 4494 NT 12 NT NT 450 4.2 NT 6.8 NT NT 451 31 280 inactive NT NT 452 73 NT250 NT NT 455 27 NT 25 NT NT 456 27 NT 19 NT NT 457 14 NT 1900 NT NT 45927 NT 14 NT NT 460 14 NT 13 NT NT 461 3.7 NT 2.7 0.075 NT 462 2.7 NT 15NT NT 470 3.5 NT NT NT NT 471 inactive NT inactive NT NT 472 2.5 NT NTNT NT 473 20 NT NT NT NT 474 4.4 NT NT NT NT 475 4.1 NT NT NT NT 476 2.3NT NT NT NT 477 2.7 1.4 NT NT NT 478 760 NT inactive NT NT 479 6 NT 15NT NT 480 3 NT NT NT NT 481 3.5 NT NT NT NT 483 2.5 NT NT NT NT 484 3.52.5 NT NT NT 485 58 NT 370 NT NT 502 100 NT inactive NT NT 511 4.1 2.514 NT NT 513 1500 NT 550 NT NT 520 6.3 NT 14 NT NT 521 570 NT 790 NT NT523 4.6 NT 11 NT NT 524 2.4 NT 10 NT NT 525 8.4 NT 11 NT NT 526 2.1 1.28.6 NT NT 529 3.3 NT 9.4 NT NT 530 190 NT 6500 NT NT 531 20 NT inactiveNT NT 532 4.1 NT 41 NT NT 533 NT NT 19 NT NT 534 no tested NT 16 NT NT542 1.7 1.2 12 NT NT 556 inactive NT 2300 NT NT 577 4.1 NT 21 NT NT 5784 NT 20 NT NT 579 6 NT 20 NT NT 582 3.5 1.6 NT NT NT 583 14 NT 50 NT NT586 6 NT 90 NT NT 588 3.3 11 120 NT NT 590 9.1 23 300 NT NT 591 38 29 NTNT NT 595 49 2.7 210 NT NT 597 2.0 1.9 58 NT NT 598 1.7 NT 35 NT NT 5991.7 NT 69 NT NT 600 11 NT 290 NT NT 601 2.4 NT 220 NT NT 602 2.04 NT 18NT NT 603 NT NT 22 NT NT 604 NT NT 10 NT NT 605 NT NT 6 NT NT 607 NT NTNT NT NT 610 NT NT NT NT NT 612 NT NT NT NT NT 613 NT NT NT NT NT 614 NTNT NT NT NT 615 NT 5.2 NT NT NT NT, not tested.

EXAMPLE 10 Activity of Bisphosphonate Compounds against Trypanosoma andLeishmania Parasites

We have obtained data for selected bisphosphonate compounds againstparasites including Trypanosoma brucei, Trypanosoma cruzi, andLeishmania major. Several compounds showed substantial activity in oneor more functional assays. See Table 5 (in some instances, the same datadepicted elsewhere herein, e.g., in Table 1, may be presented again tofacilitate comparative analysis).

TABLE 5 (6) (7) (8) (9) T. brucei L. major T. cruzi T. brucei solublevacuolar Com- FPPS FPPS IC₅₀ pyrophosphatase pound IC₅₀ (uM) Ki (nM)(ug/mL) IC₅₀ (uM) 278 0.83 18 3.07 NT 297 NT 38 NT NT 300 0.58 9 NT 35335 NT 160 NT 70 344 NT 70 NT 78 359 NT 75 NT NT 364 NT 950 NT NT 398 NT80 NT NT 443 NT 110 NT NT 444 NT 20 NT NT 445 NT 20 NT NT 446 NT 30 NTNT 447 NT 380 NT NT 449 NT NT NT NT 450 NT NT NT NT 451 NT NT NT NT 452NT NT NT NT 455 NT NT NT NT 456 NT NT NT NT 457 NT NT NT NT 459 NT NT NTNT 460 NT NT NT NT 461 0.54 50 NT NT 462 NT NT NT NT 470 0.79 NT NT NT471 NT NT NT NT 472 NT NT NT NT 473 NT NT NT NT 474 NT NT NT NT 475 NTNT NT NT 476 NT NT NT NT 477 0.89 NT NT NT 478 NT NT NT NT 479 NT NT NTNT 480 NT NT NT NT 481 NT NT NT NT 483 0.43 NT NT NT 484 1.1 NT NT NT485 NT NT NT NT 502 NT NT NT NT 511 0.76 NT NT NT 513 NT NT NT NT 5200.65 NT NT NT 521 NT NT NT NT 523 NT NT NT NT 524 0.26 NT NT NT 525 NTNT NT NT 526 0.54 NT NT NT 529 NT NT NT NT 530 NT NT NT NT 531 NT NT NTNT 532 NT NT NT NT 533 NT NT NT NT 534 NT NT NT NT 542 0.39 NT NT NT 556NT NT NT NT 577 NT NT NT NT 578 NT NT NT NT 579 NT NT NT NT 582 NT NT NTNT 583 4.6 NT NT NT 586 NT NT NT NT 588 NT NT NT NT 590 NT NT NT NT 591NT NT NT NT 595 NT NT NT NT 597 NT NT NT NT 598 0.59 NT NT NT 599 0.54NT NT NT 600 NT NT NT NT 601 2.1 NT NT NT 602 1 NT NT NT 603 0.71 NT NTNT 604 0.57 NT NT NT 605 0.29 NT NT NT 607 NT NT NT NT 610 NT NT NT NT612 NT NT NT NT 613 NT NT NT NT 614 NT NT NT NT 615 NT NT NT NT NT, nottested.Statements Regarding Incorporation by Reference and Variations

All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference, to theextent each reference is at least partially not inconsistent with thedisclosure in this application (for example, a reference that ispartially inconsistent is incorporated by reference except for thepartially inconsistent portion of the reference).

When a group of substituents is disclosed herein, it is understood thatall individual members of those groups and all subgroups, including anyisomers and enantiomers of the group members, and classes of compoundsthat can be formed using the substituents are disclosed separately. Whena Markush group or other grouping is used herein, all individual membersof the group and all combinations and subcombinations possible of thegroup are intended to be individually included in the disclosure. When acompound is described herein such that a particular isomer or enantiomerof the compound is not specified, for example, in a formula or in achemical name, that description is intended to include each isomer andenantiomer of the compound described individually or in any combination.When an atom is described herein, including in a composition, anyisotope of such atom is intended to be included. Specific names ofcompounds are intended to be exemplary, as it is known that one ofordinary skill in the art can name the same compounds differently. Everyformulation or combination of components described or exemplified hereincan be used to practice the invention, unless otherwise stated. Whenevera range is given in the specification, for example, a temperature range,a time range, or a composition range, all intermediate ranges andsubranges, as well as all individual values included in the ranges givenare intended to be included in the disclosure.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art, insome cases as of their filing date, and it is intended that thisinformation can be employed herein, if needed, to exclude (for example,to disclaim) specific embodiments that are in the prior art. Forexample, when a compound is claimed, it should be understood thatcompounds known in the prior art, including certain compounds disclosedin the references disclosed herein (particularly in referenced patentdocuments), are not intended to be included in the claim.

Where the terms “comprise”, “comprises”, “comprised”, or “comprising”are used herein, they are to be interpreted as specifying the presenceof the stated features, integers, steps, or components referred to, butnot to preclude the presence or addition of one or more other feature,integer, step, component, or group thereof.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention. It will be apparent to oneof ordinary skill in the art that methods, devices, device elements,materials, procedures and techniques other than those specificallydescribed herein can be applied to the practice of the invention asbroadly disclosed herein without resort to undue experimentation. Allart-known functional equivalents of methods, devices, device elements,materials, procedures and techniques described herein are intended to beencompassed by this invention. Whenever a range is disclosed, allsubranges and individual values are intended to be encompassed. Thisinvention is not to be limited by the embodiments disclosed, includingany shown in the drawings or exemplified in the specification, which aregiven by way of example or illustration and not of limitation.

REFERENCES

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1. A compound of formula CA1:

or a pharmaceutically acceptable salt thereof; wherein: X is H n is 1,2,or 3; R¹ and R⁵ are both hydrogens and R², R³ and R⁴ are independentlyselected from the group consisting of hydrogen, halogen, an optionallysubstituted alkyl group having 4-20 carbon atoms, an optionallysubstituted alkenyl group having 2-20 carbon atoms, an —OR group, anoptionally substituted aryl group and an optionally substitutedarylalkyl group, where R is selected from an optionally substitutedalkyl group having 1-20 carbon atoms; and R⁶ and R⁷, independently, areselected from the group consisting of a hydrogen, a halogen, a —N(R′)₂,or —SR′ group, an optionally substituted alkyl group, an optionallysubstituted alkenyl group, an optionally substituted alkoxy group, andan optionally substituted aryl group, where each R′, independently, isselected from H, an optionally substituted alkyl group and an optionallysubstituted aryl group, wherein at least one of R², R³ or R⁴ is selectedfrom the group consisting of a halogen, an optionally substituted alkylgroup having 4-20 carbon atoms, an optionally substituted alkenyl grouphaving 2-20 carbon atoms, an —OR group; and wherein R is selected froman optionally substituted alkyl group having 1-20 carbon atoms, anoptionally substituted aryl group and an optionally substituted arylalkyl group.
 2. The compound of claim 1, wherein n is
 1. 3. The compoundof claim 2, wherein R⁶ and R⁷ are both hydrogens.
 4. The compound ofclaim 1, wherein R¹ and R⁵ are both hydrogens.
 5. The compound of claim1, wherein the alkyl, alkenyl or aryl groups are optionally substitutedwith one or more halogens, —CN, —COOR″, —OR″, —COR″, —OCOOR″, —CON(R″)₂,—OCON(R″)₂, —N(R″)₂, —NO₂, —SR″, —SO₂R″, —SO₂N(R″)₂, —SOR″, alkyl,alkenyl, or aryl groups, where each R″, independently, is hydrogen, analkyl group, alkenyl group or aryl group.
 6. The compound of claim 1,wherein at least one of R², R³ or R⁴ is selected from the groupconsisting of an optionally substituted alkyl group having 4-20 carbonatoms, an optionally substituted alkenyl group having 2-20 carbon atoms,and an —OR group where R is selected from an optionally substitutedalkyl group having 1-20 carbon atoms.
 7. The compound of claim 6,wherein n is 1 and R⁶ and R⁷ are both hydrogens.
 8. The compound ofclaim 1, wherein at least one of R², R³ or R⁴ is selected from the groupconsisting of an optionally substituted alkyl group having 4-20 carbonatoms, and an —OR group where R is selected from an optionallysubstituted alkyl group having 4-20 carbon atoms.
 9. The compound ofclaim 1, wherein at least one of R², R³ or R⁴ is selected from the groupconsisting of an optionally substituted aryl group and an optionallysubstituted aryl alkyl group.
 10. The compound of claim 9, wherein thearyl groups are optionally substituted with one or more halogens, alkylgroups, alkoxy groups or hydroxy groups.
 11. The compound of claim 9,wherein at least one of R², R³ or R⁴ is selected from the groupconsisting of an optionally substituted phenyl group and an optionallysubstituted phenylmethyl group.
 12. The compound of claim 11, wherein nis 1 and R⁶ and R⁷ are both hydrogen.
 13. The compound of claim 1,wherein at least one of R², R³ or R⁴ is selected from the groupconsisting of an —OR group where R is selected from an optionallysubstituted alkyl group having 4-20 carbon atoms.
 14. The compound ofclaim 13, wherein n is 1 and R⁶ and R⁷ are both hydrogen.
 15. Thecompound of claim 1, wherein at least one of R², R³ or R⁴ is selectedfrom the group consisting of an —OR group where R is selected from analkyl group having 4-20 carbon atoms.
 16. The compound of claim 15,wherein n is 1 and R⁶ and R⁷ are both hydrogen.
 17. The compound ofclaim 1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, and R² isselected from the group consisting of optionally substituted alkylgroups having 4-20 carbon atoms, optionally substituted alkoxy groupshaving 4-20 carbon atoms and optionally substituted phenyl groups. 18.The compound of claim 17, wherein optional substitution is substitutionby one or more halogens, alkyl groups, alkoxy groups or hydroxy groups.19. The compound of claim 1, wherein n is 1, all of R¹, R³-R⁷ arehydrogens and R² is an optionally substituted alkyl group having 4-20carbon atoms.
 20. The compound of claim 1, wherein n is 1, all of R¹,R³-R⁷ are hydrogens and R² is an alkyl group having 4-20 carbon atoms.21. The compound of claim 1, wherein n is 1, all of R¹, R³-R⁷ arehydrogens and R² is an alkyl group having 4-10 carbon atoms.
 22. Thecompound of claim 1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens andR² is an alkyl group having 10-20 carbon atoms.
 23. A therapeuticcomposition comprising a compound of claim 1 and a pharmaceuticallyacceptable carrier.
 24. A pharmaceutical composition comprising acompound of claim 19 and a pharmaceutically acceptable carrier.
 25. Amethod of treating a bone resorption disorder comprising administeringto a patient in need thereof, a therapeutically effective amount of acompound of claim
 1. 26. A method of treating an infectious diseasecomprising administering to a patient in need thereof, a therapeuticallyeffective amount of a compound of claim 1 wherein the infection is dueto Trypanosoma or Leishmania parasites.
 27. A method for stimulating γδT cells which comprises the step of contacting an effective amount of acompound of claim 1 with γδ T cells.